Paternal age impairs in vitro embryo and in vivo fetal development in murine

The association between advanced paternal age and impaired reproductive outcomes is still controversial. Several studies relate decrease in semen quality, impaired embryo/fetal development and offspring health to increased paternal age. However, some retrospective studies observed no alterations on both seminal status and reproductive outcomes in older men. Such inconsistency may be due to the influence of intrinsic and external factors, such as genetics, race, diet, social class, lifestyle and obvious ethical issues that may bias the assessment of reproductive status in humans. The use of the murine model enables prospective study and owes the establishment of homogeneous and controlled groups. This study aimed to evaluate the effect of paternal age on in vitro embryo development at 4.5 day post conception and on in vivo fetal development at 16 days of gestation. Murine females (2–4 months of age) were mated with young (4–6 months of age) or senile (18–24 months of age) males. We observed decreased in vitro cleavage, blastocyst, and embryo development rates; lighter and shorter fetuses in the senile compared to the young group. This study indicated that advanced paternal age negatively impacts subsequent embryo and fetal development.

No differences were observed between groups for placental weight (0.094 ± 0.012 g and 0.092 ± 0.003 g; p = 0.90), length (0.769 ± 0.050 cm and 0.771 ± 0.021 cm; p = 0.96) and area (0.458 ± 0.072 cm 2 and Table 1. Summary of advantages for use murine as a model in scientific research.

Observations References
Anatomical and physiological similarities with humans Bryda 25 , Uhl 26 , Vandamme 27 Easily handling on laboratory animal facility Bryda 25 , Uhl 26 , Vandamme 27 Short generational interval and larger litter size Bryda 25 , Uhl 26 , Vandamme 27 Use of inbred strains promote homogenous control group on a controlled environment Casellas 28 and Barré-Sinoussi et al. 29 Full genome sequenced Waterston et al. 30 Model for male infertility O'Bryan 31 , Jamsai et al. 32  Sperm evaluation. There was no difference between the young and senile groups for the analyses performed by flow cytometry (mitochondrial membrane potential, plasma and acrosome membrane integrity, and oxidative stress; see Table 2). Considering CASA analysis, we observed lower average pathway velocity (VAP) straight-line velocity (VSL); curvilinear velocity (VCL); total sperm motility (TM), percentage of sperm with a.
Senile group b. c.    www.nature.com/scientificreports/ progressive motility (PROG), percentage of rapid sperm (RAPID) in the senile group compared to the young group, as shown on Table 2. Moreover, a higher percentage of static sperm (STATIC) was observed in the senile group compared to the young group (Table 2). No differences were observed in the other variables evaluated by CASA between the groups ( Table 2).
Correlation. We observed positive correlations between high mitochondrial membrane potential and cleavage (rho 0.88), blastocyst (rho 0.74), and embryo development rates (rho 0.77) in this study.  Table S1). When the correlation was performed only in the senile group, we observed a high positive correlation between high mitochondrial membrane potential with blastocyst (rho 0.89) and embryo development rates [(0.89-Supplementary Table S2)].

Discussion
In our study, we tested the hypothesis that the in vitro embryo and in vivo fetal development was negatively affected when senile mice are mated with reproductive-aged females. In fact, our results demonstrated that females mated with senile males presented lower cleavage, blastocyst, and embryo development rates compared to the ones mated with young males.
In the present study, we observed that senile male mice had a lower cleavage and blastocyst rate than young male mice. This data corroborates with the study performed by Katz-Jaffe et al. 37 , these authors evaluated in vitro embryo development from superovulated mice females and found that males aged 12-15 months showed a decrease in the formation of the blastocyst (71% vs. 81%) and quality of morphology (Gardner and Schoolcraft grading system) of expanded blastocyst (63% vs. 71%) when compared to male mice less than 12 month of age, showing that advanced paternal age has negative effects on embryonic development. However, authors did not report differences in cleavage rate and embryo quality in this stage.
In humans, Klonoff-Cohen et al. conducted a prospective study with 221 couples during in vitro fertilization protocol and observed that each additional year of male age was associated with an additional 11% chance of Table 2. Sperm Evaluation performed by flow cytometry and computer-assisted sperm analysis (CASA). VAP average pathway velocity, VSL straight-line velocity, VCL curvilinear velocity, ALH amplitude of lateral head displacement, BCF beat cross frequency, STR straightness, LIN linearity, TM total motility, PROG progressive motility, RAPID rapid velocity, MEDIUM medium velocity, SLOW slow velocity, STATICS non-moving sperm. Different superscript letters in each bar represent p < 0.05, as indicated by statistical T-test. www.nature.com/scientificreports/ not getting pregnant and 12% of unsuccessful births 38 . Nevertheless, Wu et al. did not observe any differences in fertilization and cleavage rates, embryo quality, and miscarriage rate when analyzing 9991 cycles of in vitro fertilization regardless of maternal (30-38 years) or paternal (30-42 years) age considered in this study 39 .
In the present experiment, we observed low embryo production rates probably due to the culture medium used in the in vitro manipulations. However, we believe that this factor did not influence the statistical results regarding the lower embryo production rates found in the senile when compared to the young males since this condition influenced both groups equally.
In this study, we observed that fetuses from females mated with senile males were lighter and smaller. It was described that men over 50 years old generate children with low birth weight in 90% of the cases, in addition to premature births 40,41 . In agreement, Katz-Jaffe et al. observed that female CF1 mice superovulated at 6-8 weeks (1-2 months) of age mated with males with more than 12 months of age presented smaller and lighter fetuses; and a decrease in placental weight 37 . Similarly, Denomme et al. verified the same changes in their study, such as the decrease in weight and length of fetuses, lighter placentas and a decrease in successful mating frequency from males' mice aged 11-15 months 42 .
Paternal age can affect placental development since changes in sperm DNA and epigenetic dysregulation can be frequent in older men 43 . Surprisingly, the diameter, area and weight of the placenta showed no statistical difference, unlike previous studies, which demonstrated that advanced paternal age affects placental development in mice 37,42 and humans 8,44 . In the human species, the placenta weight from pregnancies with older men (over 50 years old) increased compared to the group between 20 and 24 years old 44 . The consequence of the decrease in human placenta weight would be portrayed as an inadequate exchange of nutrients and gases due to the smaller surface area. On the other hand, an increase in placenta weight could indicate edema of the placental villi which would reduce the transfer of nutrients and gases 45 .
Recent studies indicate that the ratio of human newborn to placental weight may be related to perinatal changes; a higher ratio indicates insufficient oxygen to the fetus and a lower ratio suggests a suboptimal fetal condition 45,46 . In the study by Denomme et al. senile male mice were found to have a higher fetal: placental weight ratio compared to their youth 42 . Controversially, we observed in the present work that senile male mice had a lower fetal: placental weight ratio in relation to the group of young mice, which may be a consequence of the lower fetus weight observed in senile group.
Despite the differences that we observed in the embryo and fetal development, there were no differences on mitochondria function, plasmatic and acrosomal membranes integrity and oxidative stress in young and senile groups. However, we observed lower values for motility, kinetics variables, percentage of rapid sperm; and a higher percentage of static sperm in senile compared to the young group. This result indicated that the sperm of senile males are slower, and probably interferes with sperm fertilization capacity. The positive correlations between the percentages of motile, progress, and rapid sperm with cleavage, blastocyst, and embryo development rates reinforce this hypothesis.
Mitochondrial membrane potential (MMP) correlates positively with sperm parameters such as motility, sperm capacitation, and fertilization 47 . Several studies support that there is a link between aging, mitochondrial dysfunction, and decreased male fertility, as well as changes in fatty acid composition that can alter the fluidity of the inner mitochondrial membrane. In this study we observed a positive correlation between high mitochondrial membrane potential and embryo development rates in the senile group indicating that an increase in the number of sperm with high mitochondrial membrane potential may improve in vitro embryo development rates.
Controversially, Katz-Jaffe et al. 37 observed no differences in sperm motility in male mice aged 15 months and 8-10 weeks (1-2 months) of age, this result could be explained by the age of the animals used, which were 3 months younger compared to the age of senile male mice used in the present work. According to Dutta and Segunpta et al., every 9125 days of a mouse represents 1 year for men 34 . Therefore, several changes can occur within a relatively short time, such as 3 months. Moreover, Katz-Jaffe et al. used conventional microscopy to assess sperm motility 37 , and we assessed the sperm motility through the CASA system, obtaining greater accuracy and details on sperm movement pattern compared to routine evaluations by light microscopy 48 .
Results of the present study indicate that senile males present a decrease in reproductive performance. In consonance, in humans, paternal age can be associated with increased prevalence of comorbid conditions of urological character (decrease sperm motility, percentage of normal sperm morphology, sperm concentration, and increased sperm DNA fragmentation and ejaculatory dysfunction) [49][50][51] , which affect reproductive potential, fertility 46 , low conception rates to poor offspring health 52 .
The increase in paternal age compromises the motility, velocity, and coordination of sperm, and negatively influences in vitro embryo development rates and the size of the fetus in mice. Therefore, more studies are necessary to indicate when male murine reproductive senility occurs, and clarify the biological mechanisms involved in the influence of paternal age on embryo and fetal development.

Methods
The present study was conducted following ethical directives for animal experiments, complied with ARRIVE guidelines 53  www.nature.com/scientificreports/ maintained in mini-isolators (ALESCO ® , Sao Paulo, Brazil) at 22-24 °C controlled air temperature, 12 light/12 dark cycle light on room, and offered industrial pellet food and filtered water "ad libitum". Male animals were divided into 2 groups, according to their age. The young group was composed of 4-6 months old mice, corresponding to men of approximately 20 years old 34 . The senile group was composed of 18-24 months old animals, corresponding to men of approximately 60-79 years old 34 . As an inclusion factor, we used only senile murine males that did not present neurological, locomotion, ophthalmological, dermatological alterations and visible increases in body volume (tumor or edema), which may be related to metabolic disorders. Before mating, the males remained in groups of up to 4 animals in each mini-isolator, and after mating the males were isolated for the continuation of the other experiments.
We used sexually mature female mice at 2-4 months of age. The females were distributed randomly in the experimental groups and the total number of the experimental units were described in each experiment. During the evaluation of embryo and fetal development, researchers were blinded to the experimental group.
Murine female management. Females estrous were synchronized with intraperitoneal injection of 5-2.5 IU of eCG (Equine Chorionic Gonadotropin, Novormon, Zoetis, Brazil) followed by 5-2.5 IU of hCG after 48 h, approximately 1 h before the start of the Laboratory Animal Facility dark cycle (12 h).
For the embryo development experiment, we used monogamous mating (1 male:1 female). In the fetal development experiment, monogamous and polygamous (1 male:2 females) mating was performed. For all experiments, after hCG administration, females were transferred to male cage throughout the night, and mating was evaluated in the morning of the following day by visualization of the vaginal plug. Regardless of plug visualization www.nature.com/scientificreports/ all females were euthanized at the 1 th and 16 th day of gestation for in vitro embryo and fetal development, respectively.
Euthanasia procedure. Cervical dislocation was performed in pregnant females after the anesthesia procedure with Isoflurane (BioChimico, Itatiaia, Rio de Janeiro and Cristália ® , Itapira, Sao Paulo). In the males, cervical dislocation 48 was performed with no anesthesia to minimize possible seminal alterations. In a parallel experiment (data not showed) we verified sperm aggregation and agglutination when isoflurane was used for the euthanasia procedure.
Reproductive performance. We calculated the mating rate (number of females with vaginal plug/total number of females that copulated × 100) 55 per experimental group.
In vitro embryo development. Experimental groups included 7 males in the senile group and 6 males in the young group, those males were mated monogamously with 13 hormonally synchronized females (2-4 months old). Vaginal plug was not observed in two females of the senile group, with consequent absence of embryos and further embryo development. Therefore, we used 6 hormonally synchronized females for young group and 5 females for senile group, providing 9 degrees of freedom for residue in statistical analyses. The females were euthanized at 1 th day of gestation, the reproductive system was accessed according to Nagy et al. 54 49 . The presumptive zygotes were released from the oviduct after washing with HH and exposed to a 0.1% hyaluronidase solution in phosphate buffered saline (PBS) to remove cumulus cells, and washed two times in HH plus 5% fetal calf serum (FCS) and then in KSOM medium (MR 020P-5D, Millipore, Massachusetts, USA). Zygotes were IVC in 30 µl drops of KSOM medium, covered with 1-2 ml mineral oil, at 38.5 °C, 5% CO 2 , 5% O 2, and 90% N 2 , under high humidity, for 4.5 days.
On day 1.5 of IVC, we assessed cleavage rate (number of cleaved embryos/number of total structures × 100). On day 4.5, blastocyst rate including early blastocyst, expanding blastocyst, expanded blastocyst, hatching blastocyst, and hatched blastocyst (number of blastocysts/number of total structures × 100) and embryo development rate (number of blastocysts/number of cleaved embryos × 100) were assessed. Embryo evaluations were performed in stereomicroscopy (Olympus SZ61, Olympus ® , Tokyo, Japan) under 60× magnification.
Fetal development. For fetal development assessment, experimental groups included 20 senile males and 18 young, which were monogamously and polygamously mated with 43 hormonally synchronized females (2-4 months old), 22 females for the senile group and 21 females for young group. Vaginal plug was observed in 10 females mated with senile males and 17 mated with young males. At 16th day of gestation, females mated with senile and young males were euthanized to evaluate the number of total structures, viable fetuses, resorption sites, length and weight of the fetuses, and the area, length and weight of the placenta per male. The litter average from each dependent variable per male was used to perform the statistical analysis. We considered only 5 litters from senile males and 9 litters from young males, therefore 15 senile males and 9 young males did not generate litters. The experiments were conducted with 14 experimental units.
Females were euthanized 16 days after vaginal plug detection. The female reproductive tract was accessed as described previously. Hysterectomy (technique adapted from Olson and Bruce 56 ) was performed and the uterus was placed in a 35 mm Petri dish. Uterine horns were sectioned longitudinally, and the content examined. The number of total structures (viable fetus plus resorption sites), viable fetus (compatible with gestational age, E16.5) 57 and resorption sites were recorded. Fetuses and their extraembryonic tissues were removed, weighed on a digital analytical balance (model AG245, Marshall Scientific®, Hampton, USA), and the weight of fetal and placental ratio.
Photos of fetuses and placentas were taken using a megapixel digital color camera (Olympus LC30, Olympus ® , Munster, Germany) attached to a stereomicroscope (Olympus SZ61, Olympus ® , Tokyo, Japan) to measurement of fetal length (crown to rump) and placental length and area were performed by ImageJ Software (Image Processing and Analysis in Java version 1.52 j, public domain, National Institutes of Health, USA) and CellSens ® Software (Olympus Live Science ® , Olympus ® , Tokyo, Japan).

Sperm evaluation.
Five animals of the senile group and four animals of the young group were randomly selected for the semen evaluation, providing 7 degrees of freedom for residue used in statistical analysis. We performed the power analysis (PROC POWER, SAS System for Windows 9.3) in the sperm variables (CASA and cytometry) to decide the minimal number of the experimental units to provide a power higher than 80%.
Sperm was collected from epididymis cauda and vas deferens according to Yamashiro et al. 58 and Kishikawa et al. 59  www.nature.com/scientificreports/ according to Castro et al. 60 . For being a metachromatic probe, mitochondria with low and medium potential fluoresce on green and with high potential fluoresces in red. In a dark room, 0.5 µL of JC-1 (1 µM final concentration) was added to 7.5 µL with 17.5000 sperm and 30 µL of CZB-Hepes medium. Samples were analyzed by flow cytometry after 5 min' incubation. Sperm plasma membrane and acrosome integrity was evaluated by flow cytometry using propidium iodide (PI; 0.5 mg/mL, 0.9% NaCl v/v) and fluorescein isothiocyanate conjugated with Pisum sativum agglutinin (FITC-PSA; 100 µg/mL, sodium azide solution at 10%) according to Hamilton et al. 61 . Propidium iodide (PI) fluoresces when it binds to DNA, however, it only penetrates the cell when the membrane is damaged, thus indirectly revealing MP damage, emitting the red fluorescence. While PSA has specificity to acrosome membrane glycoproteins, when conjugated to FITC it marks the damaged acrosome in yellowish-green fluorescence. In a dark room, FITC-PSA solution was prepared to add 190 µL of sodium azide solution at 1% and 10 µL of FITC (final concentration 24.3 µg/mL), then 11.3 µL of PI (final concentration 6.87 µg/mL) was added to solution. About 175,000 sperm were incubated with 13 µL of this solution and 30 µL of CZB-Hepes. Samples were analyzed by flow cytometry after 5 min of incubation. This association of probes separates four sperm populations: intact membrane and intact acrosome (IMIA), intact membrane and damaged acrosome (IMDA), damaged membrane and intact acrosome (DMIA), damaged membrane, and damaged acrosome (DMDA).
The fluorescent probe CellROX green ® (Molecular Probes, Eugene, OR, USA) was used to evaluate oxidative stress. CellROX green ® quantifies intracellular Reactive Oxygen Species (ROS) when the oxidation occurs, and subsequent binding to DNA, emitting a more intense green fluorescence. According to de Castro et al., in a dark room, 0.6 µL CellROX green ® (final concentration 5 µM) was added to 60 µL of CZB-Hepes. About 175,000 sperm were incubated with 1.85 µL of this solution, after 20 min of incubation was added 0.7 µL of PI (final concentration 6.87 µg/mL) 60 . Samples were analyzed by flow cytometry after 10 min of incubation.
The mean of these scans was used for statistical analysis on Hamilton Thorne IVOS Ultimate 12. Statistical analysis. Statistical analysis was performed using the Statistical Analysis System 9.3 software (SAS Institute, Cary, NC, USA). The samples were tested to the normality of residues and homogeneity of variances. We performed the T-test procedure for independent variables. A correlation test (Spearman) was performed between sperm traits, embryo development in vivo, and fetal development, considering or not the experimental groups. The probability (p) values will be presented along with the results topic for each variable, considering p significance less than 0.05, to reject the null hypothesis. The data are presented as mean ± SEM (standard error of the mean). www.nature.com/scientificreports/